Skip to main content

Thrombin Inhibitors from Haematophagous Animals

  • Chapter
  • First Online:
Toxins and Hemostasis

Abstract

To facilitate the feeding of blood, hematophagous animals use pharmacologically active molecules to ensure free-flowing of blood at both the feeding site and within the digestion system. A number of these exogenous anticoagulants have been identified and characterized in recent years. Among them, inhibitors of thrombin form the largest group both in terms of number and diversity. To date, more than 30 different exogenous thrombin inhibitors from hematophagous animals, grouped into at least 15 unique structural classes, have been reported in the literature. In this chapter, we discuss five representative classes in detail with insights from their high resolution structures in complex with thrombin.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Notes

  1. 1.

    Superscripted prefixes ‘T’, ‘H’, ‘M’, ‘B’, ‘O’, ‘R’, ‘A’ and ‘V’ were used to indicate residues of thrombin, hirudin, haemadin, boophilin, ornithodorin, rhodniin, triabin and variegin, respectively.

References

  • Balashov, Y., 1984. Interaction between blood-sucking arthropods and their hosts, and its influence on vector potential. Annu. Rev. Entomol. 29, 137–156.

    Article  PubMed  Google Scholar 

  • Betz, A., Hofsteenge, J., Stone, S.R., 1991. Role of interactions involving C-terminal nonpolar residues of hirudin in the formation of the thrombin-hirudin complex. Biochemistry 30, 9848–9853.

    Article  PubMed  CAS  Google Scholar 

  • Betz, A., Hofsteenge, J., Stone, S.R., 1992. Interaction of the N-terminal region of hirudin with the active-site cleft of thrombin. Biochemistry 31, 4557–4562.

    Article  PubMed  CAS  Google Scholar 

  • Bode, W., Turk, D., Karshikov, A., 1992. The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human α-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1, 426–471.

    Article  PubMed  CAS  Google Scholar 

  • Braun, P.J., Dennis, S., Hofsteenge, J., Stone, S.R., 1988. Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin. Biochemistry 27, 6517–6522.

    Article  PubMed  CAS  Google Scholar 

  • Campos, I.T., Amino, R., Sampaio, C.A., Auerswald, E.A., Friedrich, T., Lemaire, H.G., Schenkman, S., Tanaka, A.S., 2002. Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas’ disease vector: gene cloning, expression and characterization of the inhibitor. Insect Biochem. Mol. Biol. 32, 991–997.

    Article  PubMed  CAS  Google Scholar 

  • Champagne, D.E., 2004. Antihemostatic strategies of blood-feeding arthropods. Curr. Drug Targets Cardiovasc. Haematol. Disord. 4, 375–396.

    Article  PubMed  CAS  Google Scholar 

  • Clore, G.M., Sukumaran, D.K., Nilges, M., Zarbock, J., Gronenborn, A.M., 1987. The conformations of hirudin in solution: a study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics. EMBO J. 6, 529–537.

    PubMed  CAS  Google Scholar 

  • Davidson, C.J., Hirt, R.P., Lal, K., Snell, P., Elgar, G., Tuddenham, E.G., McVey, J.H., 2003. Molecular evolution of the vertebrate blood coagulation network. Thromb. Haemost. 89, 420–428.

    PubMed  CAS  Google Scholar 

  • Davie, E.W., Fujikawa, K., Kisiel, W., 1991. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10363–10370.

    Article  PubMed  CAS  Google Scholar 

  • Di Cera, E., 2003. Thrombin interactions. Chest 124, 11S–17S.

    Article  PubMed  Google Scholar 

  • Dodt, J., Kohler, S., Baici, A., 1988. Interaction of site specific hirudin variants with α-thrombin. FEBS Lett. 229, 87–90.

    Article  PubMed  CAS  Google Scholar 

  • Dodt, J., Muller, H.P., Seemuller, U., Chang, J.Y., 1984. The complete amino acid sequence of hirudin, a thrombin specific inhibitor: application of colour carboxymethylation. FEBS Lett. 165, 180–184.

    Article  CAS  Google Scholar 

  • Empie, M.W., Laskowski, M., Jr., 1982. Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases. Biochemistry 21, 2274–2284.

    Article  PubMed  CAS  Google Scholar 

  • Flower, D.R., 1996. The lipocalin protein family: structure and function. Biochem. J. 318(Pt 1), 1–14.

    PubMed  CAS  Google Scholar 

  • Friedrich, T., Kroger, B., Bialojan, S., Lemaire, H.G., Hoffken, H.W., Reuschenbach, P., Otte, M., Dodt, J., 1993. A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J. Biol. Chem. 268, 16216–16222.

    PubMed  CAS  Google Scholar 

  • Greinacher, A., Warkentin, T.E., 2008. The direct thrombin inhibitor hirudin. Thromb. Haemost. 99, 819–829.

    PubMed  CAS  Google Scholar 

  • Grutter, M.G., 1994. Proteinase inhibitors: another new fold. Structure 2, 575–576.

    Article  PubMed  CAS  Google Scholar 

  • Grutter, M.G., Priestle, J.P., Rahuel, J., Grossenbacher, H., Bode, W., Hofsteenge, J., Stone, S.R., 1990. Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition. EMBO J. 9, 2361–2365.

    PubMed  CAS  Google Scholar 

  • Haruyama, H., Wuthrich, K., 1989. Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry 28, 4301–4312.

    Article  PubMed  CAS  Google Scholar 

  • Huntington, J.A., 2005. Molecular recognition mechanisms of thrombin. J. Thromb. Haemost. 3, 1861–1872.

    Article  PubMed  CAS  Google Scholar 

  • Huntington, J.A., 2008. How Na+ activates thrombin – a review of the functional and structural data. Biol. Chem. 389, 1025–1035.

    Article  PubMed  CAS  Google Scholar 

  • Isawa, H., Yuda, M., Yoneda, K., Chinzei, Y., 2000. The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway. J. Biol. Chem. 275, 6636–6641.

    Article  PubMed  CAS  Google Scholar 

  • Jackman, M.P., Parry, M.A., Hofsteenge, J., Stone, S.R., 1992. Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin. J. Biol. Chem. 267, 15375–15383.

    PubMed  CAS  Google Scholar 

  • Koh, C.Y., Kazimirova, M., Nuttall, P.A., Kini, R.M., 2009. Noncompetitive inhibitor of thrombin. Chembiochem 10, 2155–2158.

    Article  PubMed  CAS  Google Scholar 

  • Koh, C.Y., Kazimirova, M., Trimnell, A., Takac, P., Labuda, M., Nuttall, P.A., Kini, R.M., 2007. Variegin, a novel fast and tight binding thrombin inhibitor from the tropical bont tick. J. Biol. Chem. 282, 29101–29113.

    Article  PubMed  CAS  Google Scholar 

  • Koh, C.Y., Kini, R.M., 2008. Anticoagulants from hematophagous animals. Expert Rev. Hematol. 1, 135–139.

    Article  PubMed  CAS  Google Scholar 

  • Koh, C.Y., Kini, R.M., 2009. Molecular diversity of anticoagulants from haematophagous animals. Thromb. Haemost. 102, 437–453.

    PubMed  CAS  Google Scholar 

  • Lai, R., Takeuchi, H., Jonczy, J., Rees, H.H., Turner, P.C., 2004. A thrombin inhibitor from the ixodid tick, Amblyomma hebraeum. Gene 342, 243–249.

    Article  PubMed  CAS  Google Scholar 

  • Lane, D.A., Philippou, H., Huntington, J.A., 2005. Directing thrombin. Blood 106, 2605–2612.

    Article  PubMed  CAS  Google Scholar 

  • Laskowski, M., Jr., Kato, I., 1980. Protein inhibitors of proteinases. Annu. Rev. Biochem. 49, 593–626.

    Article  PubMed  CAS  Google Scholar 

  • Ledizet, M., Harrison, L.M., Koskia, R.A., Cappello, M., 2005. Discovery and pre-clinical development of antithrombotics from hematophagous invertebrates. Curr. Med. Chem. Cardiovasc. Hematol. Agents 3, 1–10.

    Article  PubMed  CAS  Google Scholar 

  • Liu, C.C., Brustad, E., Liu, W., Schultz, P.G., 2007. Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin. J. Am. Chem. Soc. 129, 10648–10649.

    Article  PubMed  CAS  Google Scholar 

  • Macedo-Ribeiro, S., Almeida, C., Calisto, B.M., Friedrich, T., Mentele, R., Sturzebecher, J., Fuentes-Prior, P., Pereira, P.J., 2008. Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick. PLoS One 3, e1624

    Article  PubMed  CAS  Google Scholar 

  • Mans, B.J., Andersen, J.F., Schwan, T.G., Ribeiro, J.M., 2008. Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: implications for the evolution of blood-feeding in the soft tick family. Insect Biochem. Mol. Biol. 38, 22–41.

    Article  PubMed  CAS  Google Scholar 

  • Mans, B.J., Louw, A.I., Neitz, A.W., 2002. Evolution of hematophagy in ticks: common origins for blood coagulation and platelet aggregation inhibitors from soft ticks of the genus Ornithodoros. Mol. Biol. Evol. 19, 1695–1705.

    Article  PubMed  CAS  Google Scholar 

  • Mao, S.J., Yates, M.T., Blankenship, D.T., Cardin, A.D., Krstenansky, J.L., Lovenberg, W., Jackson, R.L., 1987. Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech. Anal. Biochem. 161, 514–518.

    Article  PubMed  CAS  Google Scholar 

  • Markwardt, F., 1994. The development of hirudin as an antithrombotic drug. Thromb. Res. 74, 1–23.

    Article  PubMed  CAS  Google Scholar 

  • Mende, K., Petoukhova, O., Koulitchkova, V., Schaub, G.A., Lange, U., Kaufmann, R., Nowak, G., 1999. Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect. Dipetalogaster maximus cDNA cloning, expression and characterization. Eur. J. Biochem. 266, 583–590.

    Article  PubMed  CAS  Google Scholar 

  • Myles, T., Le Bonniec, B.F., Betz, A., Stone, S.R., 2001. Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I. Biochemistry 40, 4972–4979.

    Article  PubMed  CAS  Google Scholar 

  • Nienaber, J., Gaspar, A.R., Neitz, A.W., 1999. Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae). Exp. Parasitol. 93, 82–91.

    Article  PubMed  CAS  Google Scholar 

  • Noeske-Jungblut, C., Haendler, B., Donner, P., Alagon, A., Possani, L., Schleuning, W.D., 1995. Triabin, a highly potent exosite inhibitor of thrombin. J. Biol. Chem. 270, 28629–28634.

    Article  PubMed  CAS  Google Scholar 

  • Page, M.J., Macgillivray, R.T., Di Cera, E., 2005. Determinants of specificity in coagulation proteases. J. Thromb. Haemost. 3, 2401–2408.

    Article  PubMed  CAS  Google Scholar 

  • Qiu, X., Padmanabhan, K.P., Carperos, V.E., Tulinsky, A., Kline, T., Maraganore, J.M., Fenton, J.W., 1992. Structure of the hirulog 3-thrombin complex and nature of the S’ subsites of substrates and inhibitors. Biochemistry 31, 11689–11697.

    Article  PubMed  CAS  Google Scholar 

  • Ribeiro, J.M., 1995. Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect. Agents Dis. 4, 143–152.

    PubMed  CAS  Google Scholar 

  • Ribeiro, J.M., Francischetti, I.M., 2003. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu. Rev. Entomol. 48, 73–88.

    Article  PubMed  CAS  Google Scholar 

  • Richardson, J.L., Fuentes-Prior, P., Sadler, J.E., Huber, R., Bode, W., 2002. Characterization of the residues involved in the human α-thrombin-haemadin complex: an exosite II-binding inhibitor. Biochemistry 41, 2535–2542.

    Article  PubMed  CAS  Google Scholar 

  • Richardson, J.L., Kroger, B., Hoeffken, W., Sadler, J.E., Pereira, P., Huber, R., Bode, W., Fuentes-Prior, P., 2000. Crystal structure of the human α-thrombin-haemadin complex: an exosite II-binding inhibitor. EMBO J. 19, 5650–5660.

    Article  PubMed  CAS  Google Scholar 

  • Rydel, T.J., Ravichandran, K.G., Tulinsky, A., Bode, W., Huber, R., Roitsch, C., Fenton, J.W., 1990. The structure of a complex of recombinant hirudin and human α-thrombin. Science 249, 277–280.

    Article  PubMed  CAS  Google Scholar 

  • Rydel, T.J., Tulinsky, A., Bode, W., Huber, R., 1991. Refined structure of the hirudin-thrombin complex. J. Mol. Biol. 221, 583–601.

    Article  PubMed  CAS  Google Scholar 

  • Scacheri, E., Nitti, G., Valsasina, B., Orsini, G., Visco, C., Ferrera, M., Sawyer, R.T., Sarmientos, P., 1993. Novel hirudin variants from the leech Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization. Eur. J. Biochem. 214, 295–304.

    Article  PubMed  CAS  Google Scholar 

  • Scharf, M., Engels, J., Tripier, D., 1989. Primary structures of new ’iso-hirudins’. FEBS Lett. 255, 105–110.

    Article  PubMed  CAS  Google Scholar 

  • Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., Tulinsky, A., Westbrook, M., Maraganore, J.M., 1991. Structure of the hirugen and hirulog 1 complexes of α-thrombin. J. Mol. Biol. 221, 1379–1393.

    PubMed  CAS  Google Scholar 

  • Steiner, V., Knecht, R., Bornsen, K.O., Gassmann, E., Stone, S.R., Raschdorf, F., Schlaeppi, J.M., Maschler, R., 1992. Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis. Biochemistry 31, 2294–2298.

    Article  PubMed  CAS  Google Scholar 

  • Stone, S.R., Hofsteenge, J., 1986. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 25, 4622–4628.

    Article  PubMed  CAS  Google Scholar 

  • Strube, K.H., Kroger, B., Bialojan, S., Otte, M., Dodt, J., 1993. Isolation, sequence analysis, and cloning of haemadin. An anticoagulant peptide from the Indian leech. J. Biol. Chem. 268, 8590–8595.

    PubMed  CAS  Google Scholar 

  • van de Locht, A., Lamba, D., Bauer, M., Huber, R., Friedrich, T., Kroger, B., Hoffken, W., Bode, W., 1995. Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin. EMBO J. 14, 5149–5157.

    PubMed  Google Scholar 

  • van de Locht, A., Stubbs, M.T., Bode, W., Friedrich, T., Bollschweiler, C., Hoffken, W., Huber, R., 1996. The ornithodorin-thrombin crystal structure, a key to the TAP enigma? EMBO J. 15, 6011–6017.

    PubMed  Google Scholar 

  • Vitali, J., Martin, P.D., Malkowski, M.G., Robertson, W.D., Lazar, J.B., Winant, R.C., Johnson, P.H., Edwards, B.F., 1992. The structure of a complex of bovine α-thrombin and recombinant hirudin at 2.8-Ã… resolution. J. Biol. Chem. 267, 17670–17678.

    PubMed  CAS  Google Scholar 

  • Wallace, A., Dennis, S., Hofsteenge, J., Stone, S.R., 1989. Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry 28, 10079–10084.

    Article  PubMed  CAS  Google Scholar 

  • Warkentin, T.E., Greinacher, A., Koster, A., 2008. Bivalirudin. Thromb. Haemost. 99, 830–839.

    PubMed  CAS  Google Scholar 

  • Zhang, Y., Ribeiro, J.M., Guimaraes, J.A., Walsh, P.N., 1998. Nitrophorin-2: a novel mixed-type reversible specific inhibitor of the intrinsic factor-X activating complex. Biochemistry 37, 10681–10690.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by academic research grants from National University of Singapore, Singapore.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to R. Manjunatha Kini .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Springer Science+Business Media B.V.

About this chapter

Cite this chapter

Koh, C.Y., Kini, R.M. (2010). Thrombin Inhibitors from Haematophagous Animals. In: Kini, R., Clemetson, K., Markland, F., McLane, M., Morita, T. (eds) Toxins and Hemostasis. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9295-3_15

Download citation

Publish with us

Policies and ethics